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WO2013184209A1 - Mif destiné à être utilisé dans des méthodes de traitement de sujets atteints d'une maladie neurodégénérative - Google Patents

Mif destiné à être utilisé dans des méthodes de traitement de sujets atteints d'une maladie neurodégénérative Download PDF

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Publication number
WO2013184209A1
WO2013184209A1 PCT/US2013/031449 US2013031449W WO2013184209A1 WO 2013184209 A1 WO2013184209 A1 WO 2013184209A1 US 2013031449 W US2013031449 W US 2013031449W WO 2013184209 A1 WO2013184209 A1 WO 2013184209A1
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mif
polypeptide
sodl
subject
seq
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PCT/US2013/031449
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English (en)
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Adrian Israelson
Don W. Cleveland
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Ludwig Institute For Cancer Research Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present disclosure is directed to materials and methods of prophylaxis and therapy for subjects with (or at risk for) a neurodegenerative disorder, such as amyotrophic lateral sclerosis, Parkinson's Disease, Alzheimer's Disease and Huntington's Disease.
  • a neurodegenerative disorder such as amyotrophic lateral sclerosis, Parkinson's Disease, Alzheimer's Disease and Huntington's Disease.
  • ALS Amyotrophic lateral sclerosis
  • MND motor neuron disease
  • ALS is the most common adult onset motor neuron disease, affecting one in every 20,000 individuals, with an average age of onset of 50-55 years.
  • ALS is characterized by rapidly progressive degeneration of motor neurons in the brain, brainstem, and spinal cord (Cleveland et al., Nat. Rev. Neurosci., 2, 806-19, 2001). The median survival of patients from time of diagnosis is five years.
  • ALS exists in both sporadic and familial forms. Familial ALS (FALS) comprises only 5-10% of all ALS cases. Over the last decade, a number of basic and clinical research studies have focused on understanding the familial form of the disease, which has led to the identification of eight genetic mutations related to FALS.
  • Riluzole (Rilutek®, Aventis), a glutamate antagonist, currently is the only FDA-approved medication available to treat ALS. Riluzole, however, extends life expectancy by only a few months (Miller et al., Amyotrophic Lateral Sclerosis & Other Motor Neuron Disorders, 4, 191-206, 2003).
  • Creatine and a-tocopherol have shown some efficacy in relieving the symptoms of ALS in SOD1 transgenic mice, but exhibit minimal efficacy in human ALS patients (Groeneveld et al., Annals of Neurology, 53, 437-45, 2003 and Desnuelle et al., Amyotrophic Lateral Sclerosis & Other Motor Neuron Disorders, 2, 9-18, 2001).
  • insulin-like growth factor 1 transgenically (Dobrowolny et al., J Cell Biol, 168(2): 193-9, 2005) or through AAV2-delivery via intramuscular (IM) injection and subsequent retrograde axonal transport to motor nerves (Kaspar et al., Science, 301(5634):839-42, 2003).
  • IM intramuscular
  • erythropoietin Iwasaki et al., Neurol Res, 24(7):643-6, 2002
  • VEGF vascular endothelial growth factor
  • the latter is of interest because genetic analysis has implicated hypomorphic variants in the VEGF gene as a risk factor for ALS (Lambrechts et al., Nat Genet, 34(4):383- 94, 2003).
  • mice that lack hypoxia-responsive promoter elements develop a slowly progressive motor neuron disease (Oosthuyse et al., Nat Genet, 28(2): 131-8, 2001). . Subsequently, it was documented that lenti viral delivery of VEGF to the spinal cord of ALS mice delays death (Azzouz et al., Nature, 429(6990):413-7, 2004. Two independent investigators have reported that infusion of VEGF into the cerebrospinal fluid in ALS mice (Zheng et al., Ann Neurol, 56(4):564-7, 2004) and rats (Storkebaum et al., Nat Neurosci, 8(1): p. 85-92, 2005) also slow the disease course.
  • the present invention has numerous aspects and is based in part on discoveries described herein involving macrophage migratory inhibitory factor (MIF) molecular and cell biology, including discoveries including the chaperone-like activity of MIF, and more particularly chaperone-like activity towards SOD1 protein, and more particularly towards mutant SOD1; and the effect of MIF on the association and/or accumulation of SOD1 (especially mutant SOD1) with mitochondria or other organelles in cells from the central nervous system (CNS) and other insights.
  • MIF macrophage migratory inhibitory factor
  • the invention includes materials and methods for prophylaxis and for treatment.
  • Prophylaxis is especially contemplated for subjects identified as being at elevated risk for neurodegenerative disorders, e.g., due to a predisposing genetic risk factor.
  • a method of treatment comprising administering a composition to a mammalian subject at risk for, or having, a neurodegenerative disorder (including, but not limited to, Amyotrophic lateral sclerosis (ALS), Alzheimer's Disease, Parkinson's Disease and Huntington's Disease), wherein the composition comprises at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity.
  • a neurodegenerative disorder including, but not limited to, Amyotrophic lateral sclerosis (ALS), Alzheimer's Disease, Parkinson's Disease and Huntington's Disease
  • MIF macrophage inhibitory factor
  • a method of palliating a deleterious effect of mutant SOD1 in a cell comprising contacting the cell with a composition that comprises at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity in the cell.
  • MIF macrophage inhibitory factor
  • the composition is optionally administered in an amount effective to reduce accumulation of misfolded SOD1 in cells obtainable from CNS of the subject.
  • the composition is administered to a mammalian subject identified as having a familial or genetic increased risk for the neurodegenerative disorder, in a prophylactically effective amount.
  • the composition is administered to a mammalian subject with the neurodegenerative disorder, in a
  • the agent for use in the methods described herein comprises at least one substance selected from the group consisting of:
  • polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the polypeptide exhibits chaperone activity towards a mutant SOD1 protein;
  • polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11, wherein the polypeptide exhibits chaperone activity towards a mutant SOD1 protein;
  • polypeptide comprising a MIF mutant, wherein the polypeptide exhibits chaperone activity towards a mutant SOD1 protein
  • (k) a variant of any one of (a)-(j), wherein the proline corresponding to position 2 of SEQ ID NO: 2 is deleted or replaced with another amino acid, and wherein the polypeptide exhibits chaperone activity towards a mutant SOD1 protein;
  • a polynucleotide comprising, or consisting essentially of, or consisting of, a nucleotide sequence that encodes the polypeptide of any one of (a)-(k), optionally attached to one or more heterologous coding sequences or non-coding sequences (such as expression control sequences);
  • a vector especially an expression vector, comprising the polynucleotide of (1);
  • the agent comprises a polynucleotide encoding a
  • the agent comprises a polynucleotide encoding a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 9, a variant of SEQ ID NO: 2 in which Cys60 has been replaced with a serine. It should be understood that replacement with other amino acids, or deletion of the cysteine, also is contemplated.
  • the agent comprises an oligonucleotide that increases MIF expression of activity in cells from CNS.
  • the oligonucleotide in some embodiments, is an antisense oligonucleotide that binds to a nucleotide sequence that inhibits MIF expression in cells from CNS, thereby upregulating expression of the MIF protein in the CNS.
  • the antisense oligonucleotide is at least partly complementary to the microRNA-451 sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • the agent comprises a CNS precursor cell transformed or transfected with a polynucleotide that encodes a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the polypeptide exhibits chaperone activity towards a mutant SODl protein.
  • the cell is optionally isolated from CNS of the mammalian subject, wherein the transforming or transfecting is performed ex vivo, and wherein the cell or its progeny are re-administered to the same subject after the transforming or transfecting.
  • the cell is isolated from one mammalian subject and transplanted into the CNS of a different mammalian subject of the same species.
  • the cell is expanded after the transforming or transfecting, and wherein progeny cells are re-administered to the subject after the expanding.
  • the agent comprises a compound, such as a small molecule, that increases MIF chaperone activity in CNS cells, thereby upregulating expression of the MIF protein in CNS cells.
  • the methods described herein optionally further comprises screening a subject for a SODl mutation prior to the administering step.
  • the screening step optionally comprises assaying a biological sample (e.g., spinal fluid or cells obtainable from CNS of a subject) from the subject for evidence that the SODl mutation is present in the subject.
  • a biological sample e.g., spinal fluid or cells obtainable from CNS of a subject
  • Exemplary cells obtainable or present in the CNS of a subject include, but are not limited to, glial cells, glial cell precursors, astrocytes, oligodendrocytes, neural cells and neuronal progenitor cells.
  • the screening step optionally comprises analyzing a medical record for evidence that the SODl mutation is present in the subject.
  • the medical record comprises genomic nucleotide sequence information.
  • the assaying step optionally comprises analyzing nucleic acid from the subject for a mutation that codes for SODl 09 ⁇ or S0D1 G85R , relative to the SODl wild type sequence set forth in SEQ ID NO: 8.
  • the mammalian subject has a mutation (e.g., a missense mutation) in a superoxide dismutase 1 (SODl) gene.
  • the mutation is associated with SODl misfolding, SODl self-aggregation, or SODl association with one or more cellular structures such as mitochondria, endoplasmic reticulum or endosomes in cells from CNS of the subject.
  • Screening methods to identify small molecule modulators of MIF expression or activity are also contemplated as aspects of the invention.
  • An exemplary screening method comprises contacting a CNS cell with a test small molecule and determining the quantity of the MIF mRNA or protein as described herein.
  • the method comprises contacting a CNS cell with a test compound and determining the quantity of MIF mRNA or protein, MIF chaperone activity, and/or the quantity of decreased SODl misfolding, SODl self-aggregation, or SODl association with one or more cellular structures such as mitochondria, endoplasmic reticulum or endosomes in cells from CNS cells in the presence and absence of the test small molecule, as described herein.
  • An increased MIF chaperone activity (and/or decreased SODl misfolding, SODl self-aggregation, or SODl association with one or more cellular structures) in the presence of the test small molecule identifies the test small molecule as an agonist of MIF activity, and decreased MIF chaperone activity identifies the candidate small molecule as an antagonist of MIF activity.
  • the small molecule mitigates SODl misfolding, SODl self- aggregation or SODl association with one or more cellular structures such as mitochondria, endoplasmic reticulum or endosomes in cells from CNS.
  • small molecules are screened in a cell free assay.
  • a MIF protein and a SODl protein are contacted together in the presence and absence of a test molecule, and measureable decreases in SODl misfolding, aggregation, or association with cellular structures (if mitochondria, endoplasmic reticulum, endosomes, or other structures are included in the assay) identifies the test molecule as a molecule that beneficially modulate MIF activity.
  • the missense mutation codes for an amino acid alteration selected from the group consisting of SODl 09 ⁇ and S0D1 G85R , relative to the SODl wild type sequence set forth in SEQ ID NO: 8.
  • composition described herein can be administered by any route.
  • the composition is administered by intrathecal or intravascular administration.
  • the composition is administered to the cerebrospinal fluid of the subject.
  • the invention is a use of a composition that comprises at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity.
  • MIF macrophage inhibitory factor
  • the invention is a use of a composition for palliating a deleterious effect of a mutant SODl in a cell, wherein the composition comprises at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity in the cell.
  • MIF macrophage inhibitory factor
  • compositions described herein for use in treatment are themselves aspects of the invention also, e.g., as compositions of matter.
  • the methods optionally comprises administering a standard of care therapeutic to the subject in combination with the agent described herein.
  • the agent composition can be administered simultaneously with the other active agents, which may be in admixture with the agent or may be in a separate composition.
  • Each composition preferably includes a pharmaceutically acceptable diluent, adjuvant, or carrier.
  • the agents When the agents are separately administered, they may be administered in any order.
  • the standard of care therapeutic is selected from the group consisting of standard of care therapeutic selected from the group consisting of gabapentin (Neurontin®), Myotrophin® (Insulin-like Growth Factor 1, IGF-1), brain-derived neurotrophic factor (BDNF), BFGF, Rilutek® (riluzole), SR57746A, metal chelators (e.g., D-penicillamine), erythropoietin, VEGF, creatine, cyclosporin, CoQlO, inhibitors of tubulin/filament assembly, diazepam, and various vitamins (e.g., C, E and B).
  • gabapentin Neurorontin®
  • Myotrophin® Insulin-like Growth Factor 1, IGF-1
  • BDNF brain-derived neurotrophic factor
  • BFGF brain-derived neurotrophic factor
  • Rilutek® riluzole
  • SR57746A metal chelators (e.g., D-pen
  • a method of treatment comprising administering a composition to a mammalian subject at risk for, or having, a neurodegenerative disorder, wherein the composition comprises at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity.
  • MIF macrophage inhibitory factor
  • compositions for neurodegenerative disorder prophylaxis or therapy comprising at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity.
  • MIF macrophage inhibitory factor
  • composition further comprises a pharmaceutically acceptable excipient, adjuvant, diluents, or carrier in admixture with the agent.
  • missense mutation codes for an amino acid alteration selected from the group consisting of SODl G93A and SODl G85R , relative to the SODl wild type sequence set forth in SEQ ID NO: 8.
  • the cells obtainable from CNS of a subject are selected from the group consisting of glial cells, glial cell-precursors, astrocytes, oligodendrocytes, neural cells and neuronal progenitor cells.
  • the assaying comprises analyzing SODl protein or cell from CNS of the subject for evidence of SODl misfolding, SODl self- aggregation or SODl association with mitochondria, endoplasmic reticulum or endosomes in the cell.
  • the screening comprises analyzing a medical record for evidence that the SODl mutation is present in the subject.
  • composition is administered in an amount effective to reduce accumulation of misfolded SODl in cells obtainable from CNS of the subject.
  • polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the polypeptide exhibits chaperone activity towards a mutant SODl protein;
  • polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 11, wherein the polypeptide exhibits chaperone activity towards a mutant SODl protein;
  • polypeptide comprising a MIF mutant, wherein the polypeptide exhibits chaperone activity towards a mutant SODl protein
  • (k) a variant of any one of (a)-(i), wherein the proline corresponding to position 2 of SEQ ID NO: 2 is deleted or replaced with another amino acid, and wherein the polypeptide exhibits chaperone activity towards a mutant SOD1 protein;
  • a polynucleotide comprising a nucleotide sequence that encodes the polypeptide of any one of (a)-(k);
  • polynucleotide that comprises nucleotide sequence that encodes a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the polypeptide exhibits chaperone activity towards a mutant SOD1 protein.
  • the expression vector contains a promoter sequence operatively connected to polynucleotide that comprises a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 2.
  • the expression vector contains a promoter sequence operatively connected to a polynucleotide that comprises a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or a fragment thereof that exhibits chaperone activity towards a mutant SOD1 protein.
  • polynucleotide comprises a cDNA or fragment thereof that encodes a polypeptide or fragment thereof that exhibits chaperone activity towards a mutant SOD1 protein.
  • the agent comprises a CNS precursor cell transformed or transfected with a polynucleotide that encodes a polypeptide comprising an amino acid sequence at least 90% identical to SEQ ID NO: 2, wherein the polypeptide exhibits chaperone activity towards a mutant SOD1 protein.
  • oligonucleotide is an antisense oligonucleotide that binds to a nucleotide sequence that inhibits MIF expression in cells from CNS, thereby upregulating expression of the MIF protein in the CNS.
  • antisense oligonucleotide is at least partly complementary to the microRNA sequence of SEQ ID NO: 4 or SEQ ID NO: 5.
  • oligonucleotide comprises a nucleotide sequence that is complementary to bases 1 to 9 of SEQ ID NO: 4.
  • neurodegenerative disorder is selected from amyotrophic lateral sclerosis (ALS), Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease.
  • ALS amyotrophic lateral sclerosis
  • Alzheimer's Disease Alzheimer's Disease
  • Parkinson's Disease Parkinson's Disease
  • Huntington's Disease Huntington's Disease
  • a method of palliating a deleterious effect of mutant SODl in a cell comprising contacting the cell with a composition that comprises at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity in the cell.
  • MIF macrophage inhibitory factor
  • compositions for palliating a deleterious effect of a mutant SODl in a cell comprising at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity in the cell.
  • MIF macrophage inhibitory factor
  • administering to a subject identified from the screening as having the SODl mutation the composition, in an amount effective to palliate a deleterious effect of the SODl mutation in CNS cells of the subject.
  • a method of treating a mammalian subject having a disorder associated with accumulation of misfolded SODl in CNS cells of the subject comprising administering to the subject an effective amount of a composition that comprises at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity.
  • MIF macrophage inhibitory factor
  • the invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations defined by specific paragraphs above.
  • certain aspects of the invention that are described as a genus, and it should be understood that every member of a genus is, individually, an aspect of the invention.
  • aspects described as a genus or selecting a member of a genus should be understood to embrace combinations of two or more members of the genus.
  • FIG. 1 The cytosol determines mutant SOD1 association with mitochondria.
  • A Schematic outlining the different purification steps.
  • B Spinal cord but not liver cytosol of two different mutant SOD1 rats induces mutant SOD1 association with mitochondria, as demonstrated by immunoblotting. VDAC1 immunoblot shows comparable numbers of mitochondria in each assay. The input showing comparable amounts of cytosolic fractions is also displayed.
  • C Recombinant mutant SOD1G85R was measured for its association with mitochondria in the absence or presence of liver cytosol.
  • FIG. 1 MIF inhibits the association of mutant SOD1 with mitochondria.
  • A Schematic outlining the different steps toward MIF identification.
  • B The gel filtration fractions were measured for their activity to inhibit association of recombinant mutant SOD1 with mitochondria.
  • C MIF distribution correlates perfectly with inhibitory activity of the fractions as determine by western blot
  • D The activity of recombinant MIF was measured by immunoblot for the ability to inhibit recombinant mutant SODl association with
  • VDAC immunoblots show comparable amounts of mitochondria.
  • Figure 3 A factor present in unaffected tissues inhibits the association of mutant SODl with mitochondria.
  • Spinal cord but not liver cytosol of mutant SOD1G93A rat induces mutant SODl association with mitochondria, as demonstrated by immunoblotting.
  • the ability of liver cytosol to inhibit this association is proteinase K resistant and heat sensitive. Moreover, this activity is Ca2+-, hsp70- and hsp90- independent.
  • Cytochrome c immunoblot shows comparable numbers of mitochondria in each assay. The input showing comparable amounts of cytosolic fractions is also displayed.
  • FIG. 4 Coomassie staining shows the fraction with the simplest protein composition that was subjected to mass spectrometry analysis. The gel filtration fractions were measured for their activity to inhibit association of recombinant mutant SODl with mitochondria. Cytochrome c immunoblots show comparable amounts of mitochondria.
  • FIG. 6 Distribution of MIF expression through different mouse tissues.
  • A The level of MIF expression in different mouse tissues is shown by western blot. Ponceau staining is shown as a loading control.
  • B Immunoprecipitation of misfolded SODl using B8H10 antibody was performed from different tissues expressing varying levels of MIF. Tissue sample numbers are as indicated in Figure 6A.
  • FIG. 7 MIF inhibits the association of mutant SODl with mitochondria in a concentration dependent manner. The activity of increased concentrations of recombinant MIF was measured by immunoblot for the ability to inhibit recombinant mutant SODl association with mitochondria. VDAC immunoblots show comparable amounts of mitochondria.
  • FIG. 8 MIF inhibition of mutant SODl association with mitochondria is specific.
  • the activity of recombinant MIF, hsp27, cyclophilin-A or glutathione peroxidase was measured by immunoblot for the ability to inhibit recombinant mutant SODl association with mitochondria.
  • FIG. 9 MIF inhibits the accumulation of misfolded SODl.
  • A Recombinant hSODl wild type, hSODlG93A or hSODlG85R were subjected to immunoprecipitation using DSE2 in the absence or presence of recombinant MIF. The immunoprecipitates were immunoblotted using an SODl antibody. MIF levels in the unbound fraction are shown.
  • B MIF and
  • C MIFC60S expressed in NSC-34 motor neuron-like cells suppress misfolded SODl accumulation as determined by immunoprecipitation using B8H10 antibody. MIF levels are shown in the unbound fraction.
  • FIG. 10 MIF inhibits the association of mutant SODl with endosomal membranes in NSC-34 cells.
  • MIFC60S expressed in NSC-34 motor neuron-like cells suppress mutant SODl association with endosomal membranes in a concentration dependent manner as determined by immunoblot using anti SODl antibody.
  • Calnexin levels are shown as loading control and MIF levels are shown in the cytosolic fraction.
  • Figure 11 is a map of an AAV9 expression vector comprising MIF-IRES-GFP.
  • Figure 12 is an alignment of the amino acid sequences of MIF proteins identified in various species.
  • MIF macrophage migratory inhibitory factor
  • the chaperone-like activity of MIF inhibits or decreases accumulation of misfolded SODl and inhibits SODl aggregation with mitochondria, and other organelles including endoplasmic reticulum and endosomes (e.g., represented in the light endosomal preparation of subcellular fractionation) in CNS cells, and this beneficial effect can be achieved in CNS cells by increasing MIF in such cells.
  • composition comprising at least one agent selected from the group consisting of:
  • composition is administered in an amount effective to reduce accumulation of misfolded SODl in cells from the CNS of the subject.
  • administering shall include physically administering. In jurisdictions where compatible with laws or rules governing patent-eligible subject matter, "administering" shall further include the act of prescribing a controlled substance that a human subject self-administers or that a needed professional other than the prescribing authority physically administers.
  • Subjects "at risk for" having a neurodegenerative disorder are subjects having a SODl mutation that results in SODl misfolding, SODl aggregation and SODl mitochondrial membrane association in a cell from the CNS of the subject.
  • Subjects with a family member diagnosed with a neurodegenerative disorder or having a SODl mutation that results in SODl misfolding, SODl aggregation and SODl mitochondrial membrane association in a cell from the CNS are also considered to be at risk for the neurodegenerative disorder.
  • subjects are "at risk for” having a neurodegenerative disorder if they have a mutation in TDP-43, FUS/TLS, alpha synuclein, amyloid beta and huntintin, which is associated with misfolding or aggregation of these proteins and with development of one or more neurodegenerative disorders selected from ALS, Alzheimer's disease and Huntington's disease.
  • a subject is considered to "at risk” before the subject begins to exhibit symptoms or other signs of onset of the disorder.
  • MIF protein chaperone activity refers to the ability of the MIF protein to reduce SODl misfolding, SODl aggregation and SODl mitochondrial membrane association as demonstrated according to any of the in vitro assays described in Example 1.
  • Exemplary neurodegenerative disorders for prophylaxis or therapy according to the invention include amyotrophic lateral sclerosis (ALS) (familial or sporadic), Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease.
  • ALS amyotrophic lateral sclerosis
  • Standard medical criteria are used to identify ALS symptoms in a subject, or diagnose ALS in a subject.
  • the natural history of ALS is well documented (Munset T. L., 1992, The natural history of amyotrophic lateral sclerosis. In: Handbook of Amyotrophic Lateral Sclerosis, Smith R A (eds.), Chapter 2, pp. 39-63, Marcel Dekker, Inc.: New York, the entire disclosure of which is herein incorporated by reference).
  • the presenting symptoms of ALS include muscle wasting or weakness of the hands or legs. Occasionally, cramps and fasciculations precede the common presenting symptoms. Bulbar symptoms consisting of dysartria or dysphagia typically appear as the disease progresses, but can also be the presenting complaints in some subjects.
  • the "El Escorial" criteria for the diagnosis of ALS require: (1) the presence of (a) evidence of lower motor neuron (LMN) degeneration by clinical, electrophysiological or neuropathologic examination; (b) evidence of upper motor neuron (UMN) degeneration by clinical examination; and (c) a progressive spread of symptoms or signs within a region or to other regions as determined by history or
  • TQNE Neuromuscular Examination
  • MVIC maximum voluntary isometric contraction
  • ALSFRS ALS Functional Rating Scale
  • Standard medical criteria are use to identify or diagnose other neurodegenerative disorders in a subject.
  • such criteria include, but are not limited to Diagnostic and Statistical Manual of Mental Disorders, third edition (DSM-III) Alzheimer's Disease Diagnostic and Treatment Center (ADDTC), International Statistical Classification of Diseases, 10*Revision (ICD-IO), National Institute of Neurological Disorders and Stroke-Association Internationale pour labericht et PEnseignment en Neurosciences (NINDS-AIREN) and Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV). See Pohjasvaara et al, Stroke 2000, 31, 2952- 2957.
  • Clinical characterization of a patient as mild cognitive impairment is well within the skill of the practitioner. Such testing of a patient to elucidate such a condition involves performing a series of mental tests. The methods for clinical diagnosis are widely reviewed and are discussed in, e.g., Petersen et al., Arch. Neurol. 1999, 56, 303-308, the disclosure of which is incorporated herein by reference in its entirety.
  • CT computerized tomography
  • MRI magnetic resonance imaging
  • a method of palliating a deleterious effect of mutant SODl in a cell from CNS comprising contacting the cell with a composition that comprises at least one agent selected from the group consisting of: ( a) polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; (b) a polynucleotide comprising a nucleotide sequence that encodes the polypeptide of (a); and (c) an agent that increases endogenous MIF expression or MIF chaperone activity.
  • MIF macrophage inhibitory factor
  • the method comprises screening a mammalian subject for a SODl mutation, and administering to a subject identified from the screening as having the SODl mutation the composition, in an amount effective to palliate the deleterious effect of the SODl mutation in a cell from the CNS of the subject.
  • exemplary deleterious effects include, but are not limited to, the formation or accumulation misfolded SODl in the cell, or inhibition of mitochondrial activity by the SODl in the cell.
  • a method of treating a mammalian subject having a disorder associated with accumulation of misfolded SODl in cells from CNS of the subject comprising administering to the subject an effective amount of a composition that comprises at least one agent selected from: a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity; a polynucleotide comprising a nucleotide sequence that encodes the polypeptide; and an agent that increases endogenous MIF expression or MIF chaperone activity.
  • MIF macrophage inhibitory factor
  • Cells from CNS of a subject include, but are not limited to, glial cells, glial cell-precursors, astrocytes, oligodendrocytes, neural cells (i.e., neurons) and neuronal progenitor cells.
  • the methods described herein optionally comprise administering an inhibitor of SODl expression or activity to the mammalian subject.
  • the inhibitor of SODl expression or activity is an inhibitory nucleic acid, such as an antisense oligonucleotide that binds to a nucleotide sequence that inhibits SOD1 expression in a cell from CNS of the subject, thereby inhibiting expression of the SOD1 protein in the cell.
  • the methods described herein comprise administering the composition to a mammalian subject identified as having a familial or genetic increased risk for the neurodegenerative disorder, in a prophylactically effective amount.
  • prophylactically effective amount refers to an amount of the agent effective to delay onset of the neurodegenerative disorder.
  • the method is “prophylactic” when it contributes to a measurable delay in the onset of a disease or any one or more symptoms used to diagnose a disease, e.g., provides a measure of protection, prevention, or delay of onset of disease, or delay of onset of symptoms used to diagnose a disease in a subject.
  • prophylaxis While it often will be apparent to a subject and/or the subject's caregiver(s) that a measure of protection, prevention, or delay of onset of disease, or delay of onset of symptoms used to diagnose a disease has been achieved (e.g., based on experience with the disease in other subjects), prophylaxis also is demonstrated and quantifiable in the context of a controlled study, where a measure of protection, prevention, or delay of onset of disease, or delay of onset of disease symptoms is achieved in a group of treated subjects, compared to a group of untreated controls, for example. If a standard of care regimen for prophylaxis exists for a particular disease or condition, then the use of the agents for therapy described herein can be compared against the standard of care therapeutics in a controlled study.
  • the methods described herein comprise administering a "therapeutically effective amount.”
  • therapeutically effective amount refers to an amount that slows neurodegenerative progression or provides an improvement in an indicator of neurodegenerative disorder progression, as described below in greater detail.
  • slowing neurodegenerative disorder progression means the delay of a clinically undesirable change in one or more disabilities in an individual suffering from a neurodegenerative disorder, such as ALS, and is assessed by methods routinely practiced in the art, for example, the revised Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFSR), pulmonary function tests, and muscle strength measurements. Such methods are herein referred to as "indicators of ALS disease progression.” Such delay can be shown in a controlled study comparing the rate of progression of disease in treated subjects versus untreated controls. Slowing progression alternatively means the lessening of the severity of a clinically undesirable changes as assessed at a particular point in time, compared to untreated controls.
  • ALSFSR Amyotrophic Lateral Sclerosis Functional Rating Scale
  • An "improvement in a indicator of neurodegenerative disorder progression" as used herein refers to slowing of the rate of change in one or more of the indicators of a neurodegenerative disorder progression, such as ALS disease progression.
  • An improvement in an indicator of ALS disease progression also includes a lack of a measurable change in one or more of the indicators of ALS disease progression.
  • An improvement in an indicator of ALS disease progression additionally includes a positive change in one of the indicators of ALS disease progression described herein, such as, for example, an increase in an ALSFSR-R score. It is within the abilities of a physician to identify a slowing of disease progression in an individual suffering from ALS, using one or more of the disease assessment tests described herein. A physician may administer to the individual diagnostic tests, such as additional pulmonary function tests or muscle strength measurement tests, to assess the rate of disease progression in an individual suffering from ALS.
  • a slowing of neurodegenerative disorder progression may further comprise an "increase in survival time" in an individual suffering from the neurodegenerative disorder, e.g., ALS.
  • a physician can use one or more of the disease assessment tests described herein to predict an approximate survival time of an individual suffering from ALS.
  • a physician may additionally use the known disease course of ALS accompanied by a particular ALS mutation to predict survival time.
  • the "revised ALS functional rating scale” or "ALSFRS-R” is used by physicians and is a validated rating instrument for monitoring the progression of disability in ALS patients.
  • the ALSFRS-R includes 12 questions that ask a physician to rate his or her impression of an ALS patient's level of functional impairment in performing one of ten common tasks, for example, climbing stairs. Each task is rated on a five-point scale, where a score of zero indicates an inability to perform a task and a score of four indicates normal ability in performing a task. Individual item scores are summed to produce a reported score of between zero (worst) and 48 (best).
  • the identifying steps of the methods described herein optionally comprise screening the subject for the presence of a mutation in a superoxide dismutase (SOD1) gene prior to the administering step.
  • SOD1 superoxide dismutase
  • the term "mutation" includes addition, deletion, and/or substitution of one or more nucleotides in a SOD1 gene sequence.
  • Mutation(s) in the SODl gene are, in some embodiments, associated with SODl misfolding, SODl self-aggregation or SODl association with mitochondria in cells from CNS.
  • the mutation in the SODl gene is a missense mutation.
  • missense mutations include, but are not limited to missense mutations that result in SODl 093 A , S0D1 G37R , S0D1 G85R , S0D1 H46R , relative to the wild type SODl amino acid sequence of SEQ ID NO: 8.
  • Other SODl mutations that are associated with ALS are known in the art and are described in, for example, Battistini et al., European Neurological Journal, March 2010, the disclosure of which is incorporated herein by reference in its entirety.
  • the screening step comprises, in some embodiments, assaying a biological sample from the subject for evidence that the SODl mutation is present in the subject.
  • Suitable biological samples include any tissue or fluid that contains a nucleic acid or SODl protein from the subject including, but are not limited to, blood, cells from CNS (including but not limited to, glial cells, glial cell-precursors, astrocytes, oligodendrocytes, neural cells and neuronal progenitor cells) and spinal fluid.
  • the assaying comprises analyzing nucleic acid from the subject for a mutation that codes for S0D1 G93A , S0D1 G37R , S0D1 G85R , and/or S0D1 H46R , relative to the wild type SODl amino acid sequence of SEQ ID NO: 8.
  • nucleic acid of a subject is intended to include nucleic acid obtained directly from the mammalian subject (e.g., DNA, or RNA obtained from a biological sample such as spinal fluid or cells from CNS, such as neural cells isolated from spinal fluid); and also nucleic acid derived from nucleic acid obtained directly from the subject.
  • RNA derived from a biological sample from a subject complementary to RNA derived from a biological sample from a subject, and for amplifying (e.g., via polymerase chain reaction (PCR)) DNA or RNA derived from a biological sample obtained from a subject.
  • PCR polymerase chain reaction
  • Any such derived polynucleotide which retains relevant nucleotide sequence information of the subject's own DNA/RNA is intended to fall within the definition of "nucleic acid of a subject" for the purposes of the present invention.
  • the assaying comprises analyzing SODl protein expressed in the sample or a cell from the CNS of the subject for evidence of SODl misfolding, SODl self-aggregation or SODl association with mitochondria in the cell. Such evidence of aberrant protein behavior is scored as evidence that a mutation is present. Evidence of a protein mutation also can be detected by using an antibody that preferentially recognizes an epitope of a mutant SODl compared to wild type SODl. Exemplary antibodies that preferentially recognize an epitope of a mutant SOD1 compared to wild type SOD1, include, but are not limited to B8H10 and C4F6 (both of which are commercially available from Medimabs).
  • Genetic diagnosis of a mutation in a SOD1 gene can be performed using any technologies for assaying DNA for a mutation.
  • the nucleic acid sequence data can be obtained by any means known in the art.
  • the assaying step may involve any techniques available for analyzing nucleic acid to determine its characteristics, including but not limited to well-known techniques such as single-strand conformation polymorphism analysis (SSCP) (Orita et al., Proc Natl. Acad. Sci.
  • SSCP single-strand conformation polymorphism analysis
  • DPLC denaturing high pressure liquid chromatography
  • CDGE clamped denaturing gel electrophoresis
  • DGGE denaturing gradient gel electrophoresis
  • mobility shift analysis restriction enzyme analysis
  • CMC chemical mismatch cleavage
  • RNase protection assays and use of polypeptides that recognize nucleotide mismatches, such as E. coli mutS protein; and allele- specific PCR.
  • the screening comprises analyzing a database or other record (e.g., a medical record) for evidence that a SODl mutation is present in the genome of the subject.
  • a database or other record e.g., a medical record
  • Exemplary medical records include partial or complete genome sequencing records or single nucleotide polymorphism (SNP) analysis.
  • the assaying optionally involves sequencing of nucleic acid to determine nucleotide sequence thereof, using any available sequencing technique.
  • any available sequencing technique See, e.g., Sanger et al., Proc. Natl. Acad. Sci. (USA), 74: 5463-5467, 1977 (dideoxy chain termination method); Mirzabekov, TIBTECH, 12: 27-32, 1994 (sequencing by hybridization); Drmanac et al., Nature Biotechnology, 16: 54-58, 1998; U.S. Patent No.
  • the analysis may involve a determination of whether an individual possesses a particular gene allelic variant, in which case sequencing of only a small portion of nucleic acid— enough to determine the sequence of a particular codon or codons characterizing the allelic variant— is sufficient.
  • This approach is appropriate, for example, when assaying to determine whether one family member inherited the same allelic variant that has been previously characterized for another family member, or, more generally, whether a person's genome contains an allelic variant that has been previously characterized and correlated with a neurodegenerative disorder.
  • the assaying optionally comprises performing a hybridization assay to determine whether nucleic acid from the subject has a nucleotide sequence identical to or different from one or more reference sequences.
  • the hybridization involves, in some embodiments, a determination of whether nucleic acid derived from the subject will hybridize with one or more oligonucleotides, wherein the oligonucleotides have nucleotide sequences that correspond identically to a portion of the gene sequence, or that correspond identically except for one mismatch, insertion, or deletion.
  • the hybridization conditions are selected to differentiate between perfect sequence complementarity and imperfect matches differing by one or more bases.
  • Such hybridization experiments thereby can provide single nucleotide polymorphism sequence information about the nucleic acid from the human subject, by virtue of knowing the sequences of the oligonucleotides used in the experiments.
  • nucleic acid derived from the subject is subjected to gel electrophoresis, usually adjacent to one or more reference nucleic acids.
  • the nucleic acid from the subject and the reference sequence(s) are subjected to similar chemical or enzymatic treatments and then electrophoresed under conditions whereby the
  • polynucleotides will show a differential migration pattern, unless they contain identical sequences.
  • the polynucleotide sequences encoding the gene protein product may be used in hybridization or PCR assays of fluids (e.g., spinal fluid) to detect expression of the appropriate protein.
  • Such methods may be qualitative or quantitative in nature and may include Southern or northern analysis, dot blot or other membrane-based technologies; PCR technologies; dip stick, pin, chip and ELISA technologies. All of these techniques are well known in the art and are the basis of many commercially available diagnostic kits.
  • variations of the invention include analysis of two, three, four, or more of the aforementioned parameters, e.g., existence of an affected genetic relative; genetic testing for mutations in the SOD1 genes; biochemical testing of gene product activity (e.g., SOD1 misfolding, SOD1 self-aggregation or SOD1 association with mitochondria in the cell from CNS); and expression level of MIF mRNA or protein, biochemical testing of MIF chaperone activity.
  • the methods described herein optionally comprise administering to the subject a standard of care therapeutic as a co-therapy for the disorder.
  • the standard of care therapeutic can be any agent or agents used by medical professionals to treat the disorder or its symptoms.
  • agents include, but are not limited to, gabapentin (Neurontin®), Myotrophin® (Insulin-like Growth Factor 1, IGF-1), brain-derived neurotrophic factor (BDNF), BFGF, Rilutek® (riluzole), SR57746A, metal chelators (e.g., D- penicillamine), erythropoietin, VEGF, creatine, cyclosporin, CoQIO, inhibitors of
  • tubulin/filament assembly tubulin/filament assembly, diazepam, and various vitamins (e.g., C, E and B).
  • vitamins e.g., C, E and B.
  • the methods described herein optionally comprise administering to the subject an agent that inhibits the expression or activity of SOD1.
  • the agent that inhibits the expression of activity of SOD1 is an antisense oligonucleotide, such as an antisense oligonucleotide described in Smith et al., J. Clin. Invest., 116:2290-2296, 2006. Suitable doses for administration of the SOD1 antisense oligonucleotide are also disclosed in Smith et al. (supra).
  • the SOD1 antisense oligonucleotide such as an antisense oligonucleotide described in Smith et al., J. Clin. Invest., 116:2290-2296, 2006. Suitable doses for administration of the SOD1 antisense oligonucleotide are also disclosed in Smith et al. (supra).
  • the SOD1 antisense oligonucleotide such as an antisense oligonucle
  • oligonucleotide is optionally administered by continuous intrathecal infusion for a period of time of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days or longer.
  • the agent for use in accordance with the methods described herein is a polypeptide that exhibits MIF protein chaperone activity.
  • the polypeptide comprises the human wild type amino acid sequence of MIF set forth in SEQ ID NO: 2, which is encoded by the nucleotide sequence of SEQ ID NO: 1.
  • MIF proteins have been identified in other species including mouse (Genbank Accession No.
  • NP_034928.1 rat (Genbank Accession No. NP_112313.1), cow (Genbank Accession No. NP_001028780.1), zebrafish (NP_001036786), pig (Genbank Accession No.
  • NP_001070681 African clawed frog (Genbank Accession No. NP_001083650) and sheep (Genbank Accession No. NP_001072123).
  • fragment of MIF refers to a polypeptide that includes a sufficient portion of the wild type MIF such that the polypeptide retains the chaperone activity for MIF that is demonstrated in Example 1.
  • the fragment optionally is attached to heterologous sequences that do not eliminate this chaperone activity. Deletion variants, described below, are examples of fragments.
  • the agent comprises a MIF fragment that comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 105 or at least 110 amino acids of SEQ ID NO: 2 wherein the fragment exhibits chaperone activity towards a mutant SOD1 protein.
  • a polypeptide for use in a method of the invention can be modified by techniques in the art.
  • exemplary modified polypeptides include, polypeptide variants and polypeptide derivatives.
  • polypeptide variant refers to a polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the wild type amino acid sequence, wherein the variant retains the chaperone biological activity of wild type polypeptide.
  • polypeptide derivative refers to a polypeptide that is covalently modified by conjugation to therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (derivatization with polyethylene glycol) or insertion or substitution by chemical synthesis of non-natural amino acids into the wild type sequence.
  • Deletion variants are polypeptides wherein at least one amino acid residue of any amino acid sequence is removed. Deletions can be effected at one or both termini of the protein, or with removal of one or more residues within (i.e. internal to) the polypeptide. Methods for preparation of deletion variants are routine in the art. See, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring Harbor Press, the disclosure of which is incorporated herein by reference in its entirety.
  • the deletion variant comprising a polypeptide in which the Met at position 1 of SEQ ID NO: 1 is deleted.
  • the polypeptide having chaperone activity towards a mutant SOD1 protein comprises amino acids 2-115 of SEQ ID NO: 2, or a fragment thereof that exhibits the chaperone activity..
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing hundreds or more residues, as well as internal sequence insertions of one or more amino acids.
  • insertional variants can be designed such that the resulting polypeptide retains the same biological properties or exhibits a new physical, chemical and/or biological property not associated with the parental polypeptide from which it was derived. Methods for preparation of insertion variants are also routine and well known in the art (Sambrook et al., supra).
  • Fusion proteins comprising a MIF polypeptide, and a heterologous polypeptide, are another variant contemplated by the invention.
  • heterologous polypeptides which can be fused to polypeptides of interest include proteins with long circulating half-life, such as, but not limited to, immunoglobulin constant regions (e.g., Fc region); marker sequences that permit identification of the polypeptide of interest; sequences that facilitate purification of the polypeptide of interest; and sequences that promote formation of multimeric proteins.
  • a receptor fragment is fused to alkaline phosphatase (AP).
  • Substitution variants are those in which at least one residue in the polypeptide amino acid sequence is removed and a different residue is inserted in its place.
  • Modifications in the biological properties of the polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • substitution variants are designed, i.e. one or more specific (as opposed to random) amino acid residues are substituted with a specific amino acid residue. Typical changes of these types include conservative substitutions and/or substitution of one residue for another based on similar properties of the native and substituting residues.
  • hydrophobic norleucine, met, ala, val, leu, ile
  • an alignment of MIF amino acid sequences from multiple species would provide guidance with respect to residues that are conserved and preferably left unchanged or with conservative substitutions, versus residues that vary between species, are less likely to contribute to critical structure, and are more readily accepting of substitution, deletion, or insertion. Modifications made outside of these conserved motifs is less likely to alter the ability of the polypeptide to exhibit chaperone activity towards a mutant SOD1 protein.
  • the MIF variant polypeptide is MIF C60S , relative to the wild type MIF amino acid sequence set forth in SEQ ID NO: 2.
  • the MIF variant polypeptide is a polypeptide devoid of cytokine activity, but retains the chaperone-like activity described herein in Example 1.
  • the polypeptide with MIF chaperone activity is a P1G-MIF mutant (SEQ ID NO: 10) that is devoid of tautomerase activity but retains partial cytokine activity based on partial interaction with CD74.
  • the P1G-MIF mutant comprises an amino acid sequence in which the proline at position 2 of SEQ ID NO: 2 is replaced with a glycine.
  • the amino acid sequence of the P1G-MIF mutant is set forth in SEQ ID NO: 13.
  • the polypeptide with MIF chaperone activity is D-dopachrome tautomerase (DDT or MIF-2) (SEQ ID NO: 11) that retains tautomerase activity and CD74 binding.
  • Variants can also be described with respect to the percent sequence identity shared with a wild type sequence.
  • the MIF variants that comprise an amino acid sequence at least 80% (or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to the amino acid sequence of SEQ ID NO: 2 or fragment thereof, wherein the polypeptide exhibits chaperone activity towards a mutant SOD1 protein are also contemplated.
  • modifications of wild type polypeptides are not intended to as being mutually exclusive. Variants are contemplated that combine two or more types of these modifications, for example, insertions, deletions, substitutions, fusions, and/or derivatives. For example, fragments of P1G-MIF or MIF C60S , or variants thereof that share amino acid sequence similarity and retain activity, are contemplated.
  • the invention embraces polynucleotides that encode any of the polypeptides with MIF chaperone activity described herein, and constructs and compositions comprising the polynucleotides, and methods/uses of them.
  • the amino acid sequence of a polypeptide defines a set of polynucleotides that encode it, and all are contemplated for practice of the invention.
  • the polynucleotide encoding a polypeptide with MIF chaperone activity is a cDNA comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the polynucleotide encoding a polypeptide with MIF chaperone activity is a genomic sequence comprising the nucleotide sequence set forth in SEQ ID NO: 3.
  • polynucleotides that hybridize under moderately stringent or high stringency conditions to the complete non-coding strand, or complement, of such polynucleotides.
  • Complementary molecules are useful as templates for synthesizing coding molecules, and for making stable double- stranded polynucleotides. Due to the well-known degeneracy of the universal genetic code, one can synthesize numerous polynucleotide sequences that encode the polypeptides described herein. All such polynucleotides are contemplated as part of the invention.
  • Such polynucleotides are useful for recombinant expression of polypeptides of the invention in vivo or in vitro (e.g., for gene therapy).
  • a genus of similar polypeptides can alternatively be defined by the ability of encoding polynucleotides to hybridize to the complement of a nucleotide sequence that corresponds to the cDNA sequence encoding the polypeptide.
  • the invention provides a polynucleotide that comprises a nucleotide sequence that hybridizes under moderately stringent or high stringency hybridization conditions to the complement of any specific nucleotide sequence of the invention, and that encodes a polypeptide as described herein that exhibits MIF protein chaperone activity (e.g., towards a SOD1 mutant protein).
  • highly stringent conditions refers to hybridization/wash conditions selected to only permit hybridization of DNA strands whose sequences are highly
  • Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide.
  • Exemplary highly stringent hybridization conditions are as follows: hybridization at 65°C for at least 12 hours in a hybridization solution comprising 5X SSPE, 5X Denhardt's, 0.5% SDS, and 2 mg sonicated non homologous DNA per 100 ml of hybridization solution; washing twice for 10 minutes at room temperature in a wash solution comprising 2X SSPE and 0.1% SDS; followed by washing once for 15 minutes at 65°C with 2X SSPE and 0.1% SDS; followed by a final wash for 10 minutes at 65°C with 0.1X SSPE and 0.1% SDS.
  • Moderate stringency washes can be achieved by washing with 0.5X SSPE instead of 0.1X SSPE in the final 10 minute wash at 65°C.
  • Low stringency washes can be achieved by using IX SSPE for the 15 minute wash at 65°C, and omitting the final 10 minute wash.
  • conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10.
  • Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guano sine/cyto sine (GC) base pairing of the probe.
  • the hybridization conditions can be calculated as described in Sambrook et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47 to 9.51.
  • the polynucleotide comprises a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any nucleotide sequence that encodes a wild-type MIF protein, fragments or variants as described herein that demonstrate MIF chaperone activity.
  • the polynucleotide optionally includes 3' untranslated sequence from a MIF gene.
  • the polynucleotides described herein optionally comprise one or more mutations in the 3' UTR of the MIF mRNA. Such mutations have been shown to enhance stability of the MIF mRNA. See Bandres et al., Clin. Cancer Res., 15:2281, 2009, the disclosure of which is incorporated herein by reference in its entirety.
  • one or more bases selected from the group consisting of 921-928 if SEQ ID NO: 3 are deleted and replaced with another amino acid.
  • the nucleotide at position 921 and 922 of SEQ ID NO: 3 are replaced with guanines
  • the nucleotide at position 923 of SEQ ID NO: 3 is replaced with a thymine
  • the nucleotide at position 924 of SEQ ID NO: 3 is replaced with an adenine
  • the nucleotide at position 925 of SEQ ID NO: 3 is replaced with cytosine
  • the nucleotide at position 926 of SEQ ID NO: 3 is replaced with thymine
  • the nucleotide at position 927 of SEQ ID NO: 3 is replaced with guanine
  • the nucleotide at position 928 of SEQ ID NO: 3 is replaced with adenine.
  • Expression vectors are useful for recombinant production of polypeptides of the invention.
  • Expression vectors of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct.
  • Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination or other integration in a host cell.
  • Preferred expression vectors of the invention also include sequences necessary for replication in a host cell. Expression vectors are discussed in more detail below under the heading "Gene Therapy.”
  • Exemplary expression control sequences include promoter/enhancer sequences, (e.g., cytomegalovirus promoter/enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41:521-530, 1985); Rous sarcoma virus promoter (Davis et al., Hum. Gene Ther., 4: 151, 1993); Tie promoter (Korhonen et al., Blood, 86(5): 1828-1835, 1995); or simian virus 40 promoter for expression in the target mammalian cells, the promoter being operatively linked upstream (i.e.
  • promoter/enhancer sequences e.g., cytomegalovirus promoter/enhancer (Lehner et al., J. Clin. Microbiol., 29:2494-2502, 1991; Boshart et al., Cell, 41:521-530,
  • the promoter is an neuronal-specific promoter, glial-cell- specific promoter or CNS cell-specific promoter.
  • Suitable promoters for use in connection with the present invention include, but are not limited to, prion protein (Prp) promoter, vesicular acetylcholine transporter (VAChT) promoter, glial fibrillary acidic protein (GFAP) promoter, CD l ib, proteolipid protein (Pip) promoter and cytomegalovirus (CMV) promoter.
  • polynucleotides of the invention may also optionally include a suitable polyadenylation sequence (e.g., the SV40 or human growth hormone gene polyadenylation sequence) operably linked downstream (i.e. 3') of the polypeptide coding sequence.
  • a suitable polyadenylation sequence e.g., the SV40 or human growth hormone gene polyadenylation sequence
  • the invention provides vectors comprising a polynucleotide of the described herein.
  • Such vectors are useful, e.g., for amplifying the polynucleotides in host cells to create useful quantities thereof, and for expressing polypeptides of the invention using recombinant techniques.
  • the vector is an expression vector wherein the polynucleotide of the invention is operatively linked to a polynucleotide comprising an expression control sequence.
  • Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating polynucleotides of the invention are specifically contemplated.
  • Expression control DNA sequences include promoters, enhancers, and operators, and are generally selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression.
  • the polynucleotide may further optionally comprise sequences whose only intended function is to facilitate large scale production of the vector, e.g., in bacteria, such as a bacterial origin of replication and a sequence encoding a selectable marker.
  • sequences whose only intended function is to facilitate large scale production of the vector, e.g., in bacteria, such as a bacterial origin of replication and a sequence encoding a selectable marker.
  • extraneous sequences are at least partially cleaved off prior to administration to humans according to methods of the invention.
  • the agent for use in the methods described herein comprises an inhibitory nucleic acid that increases MIF expression or activity in a cell from the CNS.
  • Inhibitory nucleic acids useful in the present methods and compositions include antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double- stranded RNA interference (RNAi) compounds such as siRNA compounds, molecules comprising modified bases, locked nucleic acid molecules (LNA molecules), antagomirs, peptide nucleic acid molecules (PNA molecules), and other oligomeric compounds or oligonucleotide mimetics which hybridize to at least a portion of the target nucleic acid and modulate its function.
  • RNAi RNA interference
  • the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA (stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof.
  • RNAi interference RNA
  • siRNA short interfering RNA
  • miRNA micro, interfering RNA
  • shRNA small, temporal RNA
  • shRNA short, hairpin RNA
  • small RNA-induced gene activation RNAa
  • small activating RNAs saRNAs
  • inhibitory nucleic acids please see US2010/0317718 (antisense oligos); US2010/0249052 (double-stranded ribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNA molecules); US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA); and WO2010/129746 and WO2010/040112 (inhibitory nucleic acids).
  • dsRNA double-stranded ribonucleic acid
  • LNA molecules LNA molecules
  • siRNA analogues siRNA
  • WO2010/129746 and WO2010/040112 inhibitory nucleic acids
  • the inhibitory nucleic acid in some embodiments, is an antisense oligonucleotide that binds to a nucleotide sequence that inhibits MIF expression in a cell from the CNS, thereby upregulating expression of the MIF protein.
  • the antisense oligonucleotide is at least partly complementary to the microRNA sequence of SEQ ID NO: 4 (microRNA-451).
  • Micro RNA-451 has been shown to regulate MIF production with a perfectly inverse correlation (Bandres et al., Clin. Cancer Res., 15:2281, 2009).
  • the design and delivery of antisense oligonucleotides targeting microRNA has been described in Horwich et al., Nat. Protoc, 3: 1537-1549, 2008, the disclosure of which is incorporated herein by reference in its entirety.
  • the oligonucleotide optionally comprises a nucleotide sequence that is at least 80% (or at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) identical to SEQ ID NO: 6
  • exemplary oligonucleotides differ by 0, 1, 2, 3 or 4 bases over their length relative to SEQ ID NO: 6.
  • the oligonucleotide comprises the nucleotide sequence set forth in SEQ ID NO: 6.
  • the antisense oligonucleotide described herein is complementary to and hybridizes to a target segment of microRNA-451.
  • target segment as used herein means a sequence of microRNA-451 to which one or more antisense oligonucleotides are complementary. Multiple antisense oligonucleotides complementary to a given target segment may or may not have overlapping sequences.
  • the sequence of the antisense oligonucleotide is complementary to bases 17 to 25 of SEQ ID NO: 5 (human microRNA-451 transcript), these bases represent a target segment of microRNA-451.
  • Other contemplated target segments of microRNA-451 within SEQ ID NO: 5 include, but are not limited to bases 1 to 5 of SEQ ID NO: 5, bases 1 to 10 of SEQ ID NO: 5, bases 5 to 10 of SEQ ID NO: 5, bases 10 to 15 of SEQ ID NO: 5, bases 10 to 20 of SEQ ID NO: 5, bases 15 to 20 of SEQ ID NO: 5, bases 20 to 30 of SEQ ID NO: 5, bases 25 to 30 of SEQ ID NO: 5, bases 30 to 40 of SEQ ID NO: 5, bases 35 to 40 of SEQ ID NO: 5, bases 40 to 50 of SEQ ID NO: 5, bases 45 to 50 of SEQ ID NO: 5, bases 50 to 60 of SEQ ID NO: 5, bases 55 to 60 of SEQ ID NO: 5, bases 60 to 70 of SEQ ID NO: 5, bases 65 to 70 of SEQ ID NO: 5, bases 70 to 82 of SEQ ID NO: 5 and bases 75 to 82 of SEQ ID NO: 5.
  • sequence of the antisense oligonucleotide is
  • the antisense oligonucleotide described herein comprises from about 15 to about 30 (or from about 17 to about 25, or from about 19 to about 25, or from about 15 to about 20) bases in length.
  • the antisense oligonucleotide that comprises about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 bases in length. It is understood that non-complementary bases may be included in such inhibitory nucleic acids; for example, an inhibitory nucleic acid 30 nucleotides in length may have a portion of 15 bases that is complementary to the targeted miRNA.
  • inhibitory nucleic acids useful in the present methods are sufficiently complementary to the target microRNA-451, e.g., hybridize sufficiently well and with sufficient biological functional specificity, to give the desired effect.
  • “Complementary” refers to the capacity for pairing, through base stacking and specific hydrogen bonding, between two sequences comprising naturally or non-naturally occurring (e.g., modified as described above) bases (nucleosides) or analogs thereof. 100% complementarity is not required.
  • inhibitory nucleic acids can comprise universal bases, or inert abasic spacers that provide no positive or negative contribution to hydrogen bonding.
  • Base pairings may include both canonical Watson-Crick base pairing and non- Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are complementary to thymidine- type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as such as 3-nitropyrrole or 5- nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • A adenosine-type bases
  • T thymidine- type bases
  • U uracil-type bases
  • C cytosine-type bases
  • G guanosine-type bases
  • universal bases such as such as 3-nitropyrrole or 5- nitroindole can hybridize to and are considered complementary to any A, C, U, or T.
  • Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U, or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267.
  • a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable.
  • a complementary nucleic acid sequence for purposes of the present methods is specifically hybridizable when binding of the sequence to the target MIF microRNA molecule interferes with the normal function of the targetMIF microRNA to cause a loss of activity (e.g., inhibiting MIF-associated expression with consequent up-regulation of MIF gene expression) and there is a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under conditions in which avoidance of the non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency. Exemplary hybridization conditions are discussed elsewhere herein.
  • the inhibitory nucleic acid comprises one or more modifications comprising: a modified sugar moiety, and/or a modified internucleoside linkage, and/or a modified nucleotide and/or combinations thereof. It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.
  • the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl- O-alkyl or 2'-fluoro-modified nucleotide.
  • RNA Ribonucleic acid
  • modifications include 2'-fluoro, 2'-amino and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues or an inverted base at the 3' end of the RNA.
  • oligonucleotides modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than; 2'- deoxyoligonucleotides against a given target.
  • modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with
  • phosphorothioate backbones and those with heteroatom backbones particularly CH2 -NH-O- CH2, CH, ⁇ N(CH3) ⁇ 0 ⁇ CH2 (known as a methylene(methylimino) or MMI backbone], CH2 -O-N (CH3)-CH2, CH2 -N (CH3)-N (CH3)-CH2 and O-N (CH3)- CH2 -CH2 backbones, wherein the native phosphodiester backbone is represented as O- P— O- CH,); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.
  • PNA peptide nucleic acid
  • Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 -5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Patent Nos. 3,687,808; 4,469,863;
  • Morpholino-based oligomeric compounds are also described in Dwaine A.
  • the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354- 364, 2010; the disclosures of which are incorporated herein by reference in their entireties).
  • PMO phosphorodiamidate morpholino oligomer
  • Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts; see US patent nos.
  • Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.
  • Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring.
  • a 2'-arabino modification is 2'-F arabino.
  • the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.
  • PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of gene expression via association to complementary messenger RNA.
  • ENAs ethylene-bridged nucleic acids
  • exemplary ENAs include, but are not limited to, 2'-0,4'-C-ethylene-bridged nucleic acids.
  • the modification(s) include locked nucleic acids (LNAs) (e.g., as described in International Publication No. WO 2008/043753, U.S. Pat. Nos.
  • LNAs locked nucleic acids
  • LNAs include ribonucleic acid analogues wherein the ribose ring is "locked" by a methylene bridge between the 2'-oxgygen and the 4' -carbon - i.e., oligonucleotides containing at least one LNA monomer, that is, one 2'-0,4'-C-methylene- ⁇ -D-ribofuranosyl nucleotide.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH 3 , F, OCN, OCH 3 OCH 3 , OCH 3 0(CH 2 ) n CH 3 , 0(CH 2 ) n NH 2 or 0(CH 2 )n CH 3 where n is from 1 to about 10; Ci to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF 3 ; OCF 3 ; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; SOCH 3 ; S0 2 CH 3 ; ON0 2 ; N0 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocyclo alkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA clea
  • the modification(s) include 2'-methoxyethoxy [2'-0-CH 2 CH 2 OCH , also known as 2'-0-(2- methoxyethyl)] (Martin et al, Helv. Chim. Acta, 1995, 78, 486). In some embodiments, the modification(s) include 2'-methoxy (2'-0-CH 3 ), 2'-propoxy (2'-OCH 2 CH 2 CH 3 ) and 2'-fluoro (2'-F).
  • the inhibitory nucleic acids are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.
  • beneficial properties such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target
  • Chimeric inhibitory nucleic acids may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Patent Nos. 5,013,830; 5,149,797; 5, 220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.
  • Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions.
  • nucleobase often referred to in the art simply as “base”
  • “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5- Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, isocytosine, pseudoisocytosine, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2- (aminoalklyamino)adenine or other hetero substituted alkyladenines, 2-thiouracil, 2- thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 5-propynyluracil, 8-azaguanine, 7- de
  • the modified nucleobases comprise other synthetic and natural nucleobases such as xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8- thioalkyl, 8- hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5- bromo, 5- trifluoromethyl and other 5-substituted uracils and cytosines, 7
  • a "universal" base known in the art e.g., inosine, can also be included.
  • 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁇ 0>C. (Sanghvi, in Crooke, and Lebleu, eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions.
  • nucleobases comprise those disclosed in United States Patent No.
  • 5-substituted pyrimidines 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
  • 5- methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ⁇ 0>C (Sanghvi, et al., eds, "Antisense Research and Applications," CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions, even more particularly when combined with 2'-0-methoxyethyl sugar modifications. Modified nucleobases are described in US patent nos.
  • Inhibitory oligonucleotides can be administered directly or delivered to cells by transformation or transfection via a vector, including viral vectors or plasmids, into which has been placed DNA encoding the inhibitory oligonucleotide with the appropriate regulatory sequences, including a promoter, to result in expression of the inhibitory oligonucleotide in the desired cell, as described elsewhere herein.
  • a vector including viral vectors or plasmids
  • the inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide.
  • one or more inhibitory nucleic acids, of the same or different types can be conjugated to each other; or inhibitory nucleic acids can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type.
  • moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.
  • a thioether e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
  • Acids Res., 1992, 20, 533- 538 an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49- 54), a phospholipid, e.g., di- hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl- rac-glycero-3-H- phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl.
  • a phospholipid e.g., di- hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl- rac
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim.
  • the effectiveness of the inhibitory nucleic acid may be assessed by any of a number of assays, including reverse transcriptase polymerase chain reaction or Northern blot analysis to determine the level of existing MIF mRNA, or Western blot analysis using antibodies which recognize the MIF protein, after sufficient time for turnover of the endogenous pool after new protein synthesis is repressed.
  • assays including reverse transcriptase polymerase chain reaction or Northern blot analysis to determine the level of existing MIF mRNA, or Western blot analysis using antibodies which recognize the MIF protein, after sufficient time for turnover of the endogenous pool after new protein synthesis is repressed.
  • an inhibitory nucleic acid e.g., an antisense oligonucleotide
  • MIF a nucleic acid
  • neural cells e.g., neurons
  • neuronal progenitor cells e.g., astrocytes
  • oligodendrocytes e.g., glial cells
  • Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, Va.; Zen-Bio, Inc., Research Triangle Park, NC;
  • inhibitory nucleic acids e.g., antisense oligonucleotides
  • animals to assess their ability to inhibit expression of a target nucleic acid and produce phenotypic changes. Testing may be performed in normal animals, or in
  • inhibitory nucleic acids are formulated in a pharmaceutically acceptable diluent, such as phosphate-buffered saline.
  • Administration includes parenteral routes of administration, such as intraperitoneal, intravenous, and subcutaneous, and further includes intrathecal and intracerebroventricular routes of administration.
  • Calculation of antisense oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight.
  • RNA is isolated from spinal fluid and/or neural cells and changes in MIF gene expression is measured. Changes in MIF protein expression encoded by target nucleic acids may also be measured.
  • Stimulation in the levels of expression or activity of a MIF nucleic acid can be assayed in a variety of ways known in the art.
  • target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR.
  • RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently
  • Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE- Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
  • MIF protein levels can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS).
  • Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
  • polynucleotides are used/delivered to achieve MIF expression in cells (or achieve expression of an inhibitory nucleic acid that targets microRNA-451), a process that may be termed Gene Therapy.
  • DNA may be introduced into a cell using a variety of vectors.
  • expression constructs comprising viral vectors containing the genes of interest may be adenoviral (see, for example, U.S. Patent No. 5,824,544; U.S. Patent No. 5,707,618; U.S. Patent No. 5,693,509; U.S. Patent No. 5,670,488; U.S. Patent No. 5,585,362; each
  • CNS gene therapy is an emerging treatment modality for disorders affecting the central nervous system (CNS).
  • CNS gene therapy has been facilitated by the development of viral vectors capable of effectively infecting post-mitotic neurons.
  • the CNS is made up of the spinal cord and the brain.
  • the spinal cord conducts sensory information from the peripheral nervous system to the brain and conducts motor information from the brain to various effectors.
  • preferred polynucleotides still include a suitable promoter and polyadenylation sequence as described above. Moreover, it will be readily apparent that, in these embodiments, the polynucleotide further includes vector polynucleotide sequences (e.g., adenoviral polynucleotide sequences) operably connected to the sequence encoding a polypeptide of the invention.
  • vector polynucleotide sequences e.g., adenoviral polynucleotide sequences
  • Adeno-associated virus (AAV) vectors are considered useful for CNS gene therapy because they have a favorable toxicity and immunogenicity profile, are able to transduce neuronal cells, and are able to mediate long-term expression in the CNS (Kaplitt et al. (1994) Nat. Genet. 8: 148-154; Bartlett et al. (1998) Hum. Gene Ther. 9: 1181-1186; and Passini et al. (2002) J. Neurosci. 22:6437-6446).
  • AAV vectors One useful property of AAV vectors lies in the ability of some AAV vectors to undergo retrograde and/or anterograde transport in neuronal cells.
  • Neurons in one brain region are interconnected by axons to distal brain regions thereby providing a transport system for vector delivery.
  • an AAV vector may be administered at or near the axon terminals of neurons.
  • the neurons internalize the AAV vector and transport it in a retrograde manner along the axon to the cell body.
  • AAV2 AAV serotype 2
  • lentivirus expressing silencing human Cu/Zn superoxide dismutase (SOD 1) interfering RNA retarded disease onset of amyotrophic lateral sclerosis (ALS) in a therapeutically relevant rodent model of ALS.
  • SOD 1 superoxide dismutase
  • delivery of a polynucleotide encoding a MIF protein (or an inhibitory nucleic acid described herein) is mediated by a DNA viral vector, such as an adenovirus (Ad) or adeno-associated virus (AAV).
  • Ads adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. See, e.g.,
  • AAV vectors are derived from single- stranded (ss) DNA parvoviruses that are nonpathogenic for mammals (reviewed in Muzyscka (1992) Curr. Top. Microb. Immunol., 158:97-129). Briefly, AAV-based vectors have the rep and cap viral genes that account for 96% of the viral genome removed, leaving the two flanking 145-basepair (bp) inverted terminal repeats (ITRs), which are used to initiate viral DNA replication, packaging and integration. In the absence of helper virus, wild-type AAV integrates into the human host-cell genome with preferential site- specificity at chromosome 19q 13.3 or it may be maintained episomally.
  • a single AAV particle can accommodate up to 5 kb of ssDNA, therefore leaving about 4.5 kb for a transgene and regulatory elements, which is typically sufficient.
  • trans-splicing systems as described, for example, in U.S. Pat. No. 6,544,785, may nearly double this limit.
  • AAV vectors contemplated for delivery of a polynucleotide encoding a protein described herein (or an inhibitory nucleic acid described herein) include, but are not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9.
  • the AAV vector is AAV9.
  • the expression vector comprises the nucleotide sequence set forth in SEQ ID NO: 12.
  • Pseudotyped AAV vectors are those which contain the inverted terminal repeats (ITRs) of one AAV serotype and the capsid of a second AAV serotype; for example, an AAV vector that contains the AAV2 capsid and the AAV1 ITRs (i.e. AAV 1/2) or an AAV vector that contains the AAV5 capsid and the AAV2 ITRs (i.e. AAV2/5).
  • ITRs inverted terminal repeats
  • a viral vector stock For viral vectors, one generally will prepare a viral vector stock. Depending on the kind of virus and the titer attainable, one will deliver, for example, 1 X 10 4 , 1 X 10 5 , 1 X 10 6 , 1 X 10 7 , 1 X 10 8 , 1 X 10 9 , 1 X 10 10 , 1 X 10 11 or 1 X 10 12 infectious particles to the patient, or a dose in a range defined by any two of these values. Similar figures may be extrapolated for liposomal or other non-viral formulations by comparing relative uptake efficiencies. Formulation as a pharmaceutically acceptable composition is discussed below.
  • Non-viral delivery mechanisms contemplated include calcium phosphate precipitation (Graham and Van Der Eb, Virology, 52:456-467, 1973; Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987; Rippe et al., Mol. Cell Biol., 10:689-695, 1990) DEAE- dextran (Gopal, Mol. Cell Biol., 5: 1188-1190, 1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986; Potter et al., Proc. Nat. Acad. Sci. USA, 81:7161-7165, 1984), direct microinjection (Harland and Weintraub, J.
  • the expression vector (or indeed the polynucleotides or polypeptides described herein) may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multi-lamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • the lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker, pp. 87-104, 1991).
  • the addition of DNA to cationic liposomes causes a topological transition from liposomes to optically birefringent liquid-crystalline condensed globules (Radler et al., Science, 275(5301):810-4, 1997).
  • These DNA-lipid complexes are potential non- viral vectors for use in gene therapy and delivery.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., Science, 243:375-378, 1989).
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear nonhistone chromosomal proteins (HMG-1) (Kato et al., J. Biol. Chem., 266:3361-3364, 1991).
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al., Nature, 327:70-73, 1987).
  • Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., Proc. Natl. Acad. Sci USA, 87:9568-9572, 1990).
  • the microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.
  • the invention provides host cells, including prokaryotic and eukaryotic cells, that are transformed or transfected (stably or transiently) with polynucleotides of the invention or vectors of the invention.
  • Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA
  • host cells are useful for amplifying the polynucleotides and also for expressing the polypeptides of the invention encoded by the polynucleotide.
  • the host cell may be isolated and/or purified.
  • the host cell also may be a cell transformed in vivo to cause transient or permanent expression of the polypeptide in vivo.
  • the host cell may also be an isolated cell transformed ex vivo and introduced post-transformation, e.g., to produce the polypeptide in vivo for therapeutic purposes.
  • the definition of "host cell” explicitly excludes a transgenic human being.
  • any host cell is acceptable, including but not limited to bacterial, yeast, plant, invertebrate (e.g., insect), vertebrate, and mammalian host cells.
  • invertebrate e.g., insect
  • vertebrate e.g., a host cell
  • mammalian host cells For developing therapeutic preparations, expression in mammalian cell lines, especially human cell lines, is preferred.
  • Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be desirable to confer optimal biological activity on
  • polypeptides described above that have been covalently modified to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
  • the invention provides a neuronal precursor progenitor cell transformed or transfected ex vivo with the gene(s) encoding a MIF polypeptide, and the transfected cells as administered to the mammalian subject.
  • compositions and routess of Administration may be administered in any suitable manner using an appropriate pharmaceutically acceptable vehicle, e.g., a pharmaceutically acceptable diluent, adjuvant, excipient or carrier.
  • a pharmaceutically acceptable diluent e.g., a pharmaceutically acceptable diluent, adjuvant, excipient or carrier.
  • Liquid, semisolid, or solid diluents that serve as
  • diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl and
  • propylhydroxybenzoate talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil, and cocoa butter.
  • Such formulations are useful, e.g., for administration of polypeptides or polynucleotides of the invention to mammalian (including human) subjects in therapeutic regimens.
  • an agent for use in accordance with the methods described herein is combined with one or more pharmaceutically acceptable carriers for an injectable formulation.
  • the carriers are isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, allow the constitution of injectable solutions.
  • the injectable preparations are a solution or suspension in a nontoxic parenterally acceptable solvent or diluent, such as saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures of such salts), Ringer's solution, dextrose, water, sterile water, glycerol, ethanol, and combinations thereof. 1,3-butanediol and sterile fixed oils are conveniently employed as solvents or suspending media. Any bland fixed oil can be employed including synthetic mono- or di-glycerides. Fatty acids such as oleic acid also find use in the preparation of injectables.
  • a nontoxic parenterally acceptable solvent or diluent such as saline, buffered saline, isotonic saline (e.g. monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride, or mixtures
  • the agent is, in some embodiments, targeted to the central nervous system (CNS) of an individual suffering from a neurodegenerative disorder, such as ALS, and in particular to the regions of the CNS affected by the neurodegenerative disorder.
  • CNS central nervous system
  • a neurodegenerative disorder such as ALS
  • one method of providing the agent to the tissues of the CNS is via administration of the agent directly into the cerebrospinal fluid (CSF).
  • Means for delivery to the CSF include, but are not limited to, intrathecal (IT) and intracerebroventricular (ICV) administration and lumbar puncture.
  • ⁇ or ICV administration is achieved through the use of surgically implanted pumps (e.g., an infusion pump) that infuse a therapeutic agent into the CSF.
  • ⁇ , ICV or lumbar administration of an agent described herein is for a period of about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 20 hours or about 24 hours.
  • the antisense oligonucleotide is continuously infused into the CSF for the entire course of treatment; such administration is referred to as “continuous infusion” or, in the case of IT infusion, "continuous ⁇ infusion.”
  • administering an antisense oligonucleotide to the CSF employs an infusion pump, such as Medtronic SyncroMed®II pump.
  • the SyncroMed® II pump is surgically implanted according the procedures set forth by the manufacturer.
  • the pump contains a reservoir for retaining a drug solution, which is pumped at a programmed dose into a catheter that is surgically implanted.
  • the catheter is surgically intrathecally implanted.
  • kits which contain the necessary reagents to carry out the assays or therapies described herein.
  • reagents are packaged together but not in admixture.
  • the invention provides a compartment kit to receive, in close confinement, one or more containers which comprises: (a) a first container comprising a viral vector comprising a nucleotide sequence that encodes a polypeptide that exhibits macrophage inhibitory factor (MIF) protein chaperone activity, and a promoter operably linked to the nucleotide sequence that is capable of promoting expression of the nucleotide sequence in mammalian cells; and optionally (b) a second container comprising a viral vector comprising a nucleotide sequence that is at least partly complementary to SEQ ID NO: 4 or SEQ ID NO: 5, wherein the nucleotide sequences binds to microRNA that inhibits MIF expression in cells from the CNS.
  • MIF macrophage inhibitory factor
  • a compartment kit includes any kit in which reagents are contained in separate containers.
  • Such containers include small glass containers, plastic containers or strips of plastic or paper.
  • Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross- contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another.
  • the method comprises contacting a CNS cell with a test compound and determining the quantity of the MIF mRNA or protein as described herein. In some embodiments, the method comprises contacting a CNS cell with a test compound and determining the quantity of MIF mRNA or protein, MIF chaperone activity, and/or the quantity of decreased SODl misfolding, SODl self-aggregation, or SODl association with one or more cellular structures such as mitochondria, endoplasmic reticulum or endosomes in cells from CNS cells as described herein.
  • the quantity of MIF mRNA or protein, SODl misfolding, SODl self-aggregation, or SODl association with one or more cellular structures such as mitochondria, endoplasmic reticulum or endosomes may be compared with the quantities produced in the absence of the test compound.
  • Another aspects of the invention relate to methods of screening for MIF agonist compounds which permeate the blood brain barrier.
  • Another aspect of the invention relates to methods of screening for compounds that inhibit mutant SODl from binding to mitochondria.
  • a SODl protein (which may be a mutant SODl) and mitochondria are contacted together in the presence and absence of a test molecule, and measureable decreases in the binding of mutant SODl to mitochondria identifies the test molecule as a molecule that inhibits binding of mutant SODl to mitochondria.
  • Stimulation in the levels of expression or activity of a MIF nucleic acid can be assayed in a variety of ways known in the art.
  • target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR.
  • RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art.
  • Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM® 7600, 7700, or 7900 Sequence Detection System, available from PE- Applied Biosystems, Foster City, Calif, and used according to manufacturer's instructions.
  • Quantitation of target RNA levels may be accomplished by quantitative real-time PCR using the ABI PRISM® 7600, 7700, or 7900 Sequence Detection System (PE- Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. Methods of quantitative real-time PCR are well known in the art.
  • small molecules are screened in a cell free assay.
  • a MIF protein and a SODl protein (which may be a mutant SODl) are contacted together in the presence and absence of a test molecule, and measureable decreases in SODl misfolding, aggregation, or association with cellular structures (if mitochondria, endoplasmic reticulum, endosomes, or other structures are included in the assay) identifies the test molecule as a molecule that beneficially modulate MIF activity.
  • MIF protein levels can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS).
  • Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.
  • Example 1 Demonstration that Macrophage Migration Inhibitory Factor Affects SODl Folding and has a Therapeutic Indication in Neural Cells
  • MIF Macrophage Migration Inhibitory Factor
  • SODl superoxide dismutase
  • Transgenic Rats Transgenic rats expressing hSODl wt , hSODl G93A and hSODl H46R were as originally described in Chan et al., J. Neurosci, 18:8292, 1998; Howland et al., Proc. Natl. Acad. Sci. USE, 99: 1604, 2002; and Nagai et al., J. Neurosci., 21:9246, 2001, respectively. All animal procedures were consistent with the requirements of the Animal Care and Use Committee of the University of California. [00231] Subcellular Fractionation: Mitochondria were purified as previously described (Vande Velde et al, Proc. Natl. Acad. Sci. USA, 105:4022, 2008). Tissues were
  • HB ice-cold homogenization buffer
  • Mitochondria were overlaid with an equal volume of 1.175 g/ml and 1.079 g/ml Optiprep and centrifuged at 50,000 x g for 4 hours (SW-55; Beckman). Mitochondria were collected at the 1.079/1.175 g/ml interface and washed once to remove the Optiprep. Optiprep stock solution was diluted in 250 mM sucrose, 120 mM Tris-HCl (pH 7.4), 6 mM EDTA plus protease inhibitors.
  • Liver was homogenized in 5 volumes of ice-cold homogenization buffer (HB) on ice. Homogenates were centrifuged at 1000 x g for 5 min. Supernatants were recovered, and centrifuged again at 1000 x g for 5 min. Supernatant was centrifuged at 12,000 x g for 10 min to yield a crude mitochondrial pellet. These mitochondria were resuspended in HB (without EDTA) and centrifuged again at 12,000 x g for 10 min. The pellet was resuspended in a small volume of HB without EDTA.
  • HB ice-cold homogenization buffer
  • Isolated mitochondria 100 ⁇ g were solubilized in immunoprecipitation (IP) buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 plus protease inhibitors] and incubated overnight with DSE2 or B8H10 (Medimabs) antibodies previously crosslinked to Dynabeads protein G (Invitogen) with dimethyl pimelimidate (Pierce) according to the manufacturer's instructions. The beads were magnetically isolated and washed three times with IP buffer. Samples were eluted with boiling in 2. Ox sample buffer.
  • IP immunoprecipitation
  • DSE2 Antibodies Disease-specific epitopes (DSE) of SOD1 were as previously described (Vande Velde et al., supra; Israelson et al., Neuron, 67:575, 2010). The epitope recognized by the antibodies was predicted to be exposed and unstructured during misfolding or metal depletion (Rahkit et al., Nat. Med., 13:754, 2007). Two independent IgG
  • monoclonal clones of monoclonal antibody DSE2 (3H1 and 8D1) were selected by reactivity to the DSE2 peptide (comprising the electrostatic loop of hSODl; residues 125-142 of SEQ ID NO: 8), to denatured and/or oxidized hSODl in vitro.
  • Anti-SODl Commercial antibodies include goat anti-SODl (C-17), goat anti-MIF (N- 18), monoclonal anti-MIF (Q-18), rabbit anti-CyPA (H-24), goat anti-AATC (V-14), goat anti-CTH (P-15), monoclonal anti-Profilin-1 (C-2), monoclonal anti ADH (A-8), rabbit anti- Arginase I (H-52), goat anti-hsp 27 (C-20) and monoclonal anti GPx-1/2 (D-12) from Santa Cruz Biotechnology. Sheep anti-SODl and monoclonal anti-VDAC/porin (31HL) from Calbiochem, rabbit anti calnexin (Stressgen) and rabbit anti-cytochrome c (BD Biosciences). Horseradish peroxidase-conjugated anti-mouse, anti-rabbit, or anti-goat IgG secondary antibodies (Jackson Immunochemicals) were used and detected by ECL (GE Biosciences).
  • NSC-34 cells were grown at 37 °C and 5% C02 in DMEM supplemented with 10% tetracycline-free FBS and penicillin/streptomycin. Transfection was performed using Lipofectamine-2000 (Invitrogen) according to the manufacture's protocol. The cells were collected 24 hours after transfection and analyzed for misfolded protein accumulation.
  • MS/MS data were collected by an LTQ
  • Orbitrap Discovery and subsequently searched on Sorcerer-SEQUEST using a semitryptic monoisotopic database generated for the human IPI database, version 3.47.
  • a 20-ppm parent mass tolerance and variable modification for lysine and arginine were included in the search.
  • the searched data were then analyzed by TPP.
  • MIF macrophage migration inhibitory factor
  • MIF MIF is synthesized as a soluble, cytoplasmic protein (it has no signal sequence for co-translational incorporation into the endoplasmic reticulum). Indeed, we have validated that most MIF is found in the soluble cytoplasmic supernatant of liver after 100,000 x g centrifugation to remove even small vesicles (Figure 6).
  • MIF has previously been implicated in intracellular protein chaperone activity, with switching from multimeric to monomeric forms exposing a hydrophobic surface that can provide ATP-independent chaperone activity (Cherepkova et al., supra). Curiously, this parallels the well-known ATP-dependent protein chaperone Hsp70, for which dual roles as a chaperone and cytokine have been identified (Asea et al., Nat. Med., 6:435, 2000).
  • MIF C60S - SEQ ID NO: 9 a mutant version of MIF that was shown before to lose completely its oxidoreductase activity.
  • Expression of MIF C60S in NSC-34 cells inhibits the accumulation of misfolded SODl in a dose dependent manner ( Figure 9C).
  • MIF C60S a MIF protein comprising a C60S or other mutation, wherein the MIF retains chaperone-like activity towards SODl but lacks thiol-oxidoreductase activity.
  • Example 2 Demonstration that MIF Affects Aggregation of SODl and has a Therapeutic Indication in Neural Cells
  • Example 1 The experiments described in Example 1 are repeated with other SODl variants to demonstrate that MIF has chaperone-like activity with respect to superoxide dismutase (SODl) protein mutants in addition to the SODl 09 ⁇ and S0D1 G85R tested in Example 1, and that this activity has a therapeutic indication in neural cells harboring the other SOD1 variants.
  • SODl superoxide dismutase
  • Example 3 Demonstration that MIF Mutants Affect Aggregation of SOD1 and have a Therapeutic Indication in Neural Cells
  • MIF Family members e.g., MIF2 (Merk et al., Proc. Natl. Acad. Sci. USA., early edition, pages 1- 9, 2011)
  • MIF mutants e.g., P1G-MIF (Fingerle-Rowson et al., Mol. Cell. Biol., 29: 1922- 1932, 2009
  • various N-terminal, C-terminal, or internal deletion variants of MIF have chaperone-like activity with respect to mutant SOD1 protein and that this activity has a therapeutic indication in neural cells.

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Abstract

La présente invention concerne des matériels et des méthodes de prophylaxie et de thérapie destinés à des sujets atteints (ou présentant le risque d'être atteints) d'une maladie neurodégénérative, telle que la sclérose latérale amyotrophique, la maladie d'Alzheimer, la maladie de Parkinson et la chorée de Huntington.
PCT/US2013/031449 2012-06-04 2013-03-14 Mif destiné à être utilisé dans des méthodes de traitement de sujets atteints d'une maladie neurodégénérative WO2013184209A1 (fr)

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US20170152517A1 (en) * 2014-07-31 2017-06-01 Association Institut De Myologie Treatment of amyotrophic lateral sclerosis
US11911436B2 (en) 2021-06-02 2024-02-27 The Curators Of The University Of Missouri Amphiphilic peptide chaperones and methods of use

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